Consequently, ribosomal protein (RP) and ribosome biogenesis (RiBi; including rRNA modifiers, assembly factors and subunits of RNA polymerases I and III [
78,
79]) genes are two groups of genes most strongly affected by carbon source shifts. RP and RiBi genes each form regulons, i.e., groups of genes that appear highly coordinated in their expression [
78,
79]. Approximately 116 RP genes and >200 RiBi genes exhibit a rapid increase in expression after glucose addition to glucose-depleted cultures [
79,
80,
81], while glucose-starved cells dramatically reduce the level of RP and RiBi transcripts within 30 min [
79].
3.1.1. Ribosome Biogenesis: Dot6/Tod6 and Stb3
Transcriptomic studies found that activation of the PKA pathway (e.g., via overexpression of activated Ras2 or activated Gpa2) recapitulated transcriptional changes induced by glucose addition to cultures without a fermentable carbon source for a large number of genes [
81,
82].
Conversely, the glucose-dependent transcriptional changes were largely blocked when simultaneously inhibiting PKA in one study. Despite its apparent genetic interaction with PKA, inhibition of Sch9 had little effect [
82].
This observation was seemingly in stark contrast to a previous finding that a majority of genes induced by glucose addition in a wt strain were still induced in a
tpkw strain in which cAMP-dependent regulation of PKA is disabled through
BCY1 deletion [
81]. This group of genes was strongly enriched in RiBi genes. Given its genetic interaction with PKA, TOR/Sch9 signaling was an obvious suspect for the redundant pathway [
81].
An interesting temporal perspective on the contribution of PKA and TOR signaling to RiBi transcriptional regulation has recently been unveiled: after an initial phase upon glucose addition, in which PKA was the dominant factor necessary for RiBi gene transcription, a co-operative effect between the pathways, with TOR signaling gaining importance, was observed [
83]. As inhibition of PKA and Sch9 by Zaman et al. was performed 20 min after glucose addition, much effect of PKA and little of Sch9 inhibition was observed since the PKA contribution was dominant at this timepoint [
82].
At face value, this does not resolve the discrepancy to the study by Wang et al., as both early and late induction of RiBi genes in
tpkw were observed here [
81]. Invoking the earlier proposal of indirect negative TOR–PKA interaction, it can, however, not be ruled out that the temporal dynamics of TOR signaling in response to glucose are altered in this strain in adaptation to the mutations in the PKA pathway. These effects would be less obvious in the context of instantaneous inhibition used by Zaman, 2009 [
82].
If the temporal observations by Kunkel et al. hold true for other transcript classes remains to be determined. Interestingly, as detailed in the following, the dynamics observed for RiBi genes are not adequately explained by current knowledge of their PKA- and TOR-dependent mechanisms of transcription regulation [
83].
The architectures of Ribi gene promoters are distinct from the one of RP genes, with Rap1-binding sites only present in a small subset of RiBi-promoters [
84]. Instead, RiBi-promoters are enriched in PAC (Polymerase A and C) and RRPE (rRNA processing element) motifs [
80,
81,
85,
86,
87]. The PAC and RRPE elements are each found in approximately half of the RiBi-promoters and approximately one-quarter of the promoters contain both elements [
79]. Both elements function in the binding of transcriptional repressors, Dot6 and its homolog Tod6 in the former, and Stb3 in the latter case [
24,
88]. These transcription factors exert their repressive role via recruitment of the Rpd3L histone deacetylase complex [
24]. This is in agreement with observations that Rpd3L is recruited to a number of rapamycin-repressed genes upon rapamycin treatment [
89] but in contradiction to earlier studies that found Rpd3L binding to be constitutive [
90,
91].
Double deletion of
DOT6 and
TOD6 led to a noticeable change in transcriptional repression after glucose and nitrogen starvation [
92] and both proteins were dephosphorylated as early as 5 min after these starvations and upon various stresses, such as heat, oxidative and osmotic stress, as well as upon rapamycin treatment [
93]. The same study demonstrated nuclear localization of Dot6 and Tod6 following stress, nitrogen- or glucose starvation.
It was shown recently that the levels of Dot6 and Tod6 are low when cells are grown in medium without a fermentable carbon source and only rise upon glucose addition and that early RiBi gene induction upon glucose addition occurred normally in a
dot6∆ tod6∆ strain [
83]. Therefore, the regulatory function of these transcription factors is likely relevant upon depletion of a fermentable carbon source rather than in the relief of repression upon encountering glucose. The mechanism of RiBi gene regulation in the latter transition is still unclear [
83].
One of the early phospho-proteomics screens into TOR signaling found reduced phosphorylation of Dot6 upon rapamycin treatment, which was alleviated by mutations of Sch9 that rendered it active independent of TORC1 [
94]. Similar observations were made for its paralog Tod6, but here hypo-phosphorylation was additionally alleviated by rapamycin-insensitive tap42-11. These observations were only partially reflected by gel-shift assays of Dot6, while Tod6 exhibited clear dephosphorylation, that depended on the inhibition of Sch9. Dot6 was also detected in a screen for proteins interacting with a substrate-trapping mutant of Tpk1 and subsequently shown to be phosphorylated by PKA in vitro [
95]. Later, in vitro phosphorylation of Dot6/Tod6 by Sch9, as well as a decrease in Dot6/Tod6 phosphorylation after inhibition of Sch9 or PKA in vivo was also observed [
24]. The consequence of
DOT6 and/or
TOD6 deletion for RiBi gene repression upon rapamycin treatment or analog-sensitive PKA inhibition have been evaluated, and a more prominent role was attributed to Tod6 downstream of TORC1 and to Dot6 downstream of PKA [
92].
A further transcriptional repressor, Stb3, that forms part of the RPD3L complex [
96] was also found to be phosphorylated by PKA and Sch9 in vitro [
24,
97]. Stb3 phosphorylation also decreased upon Sch9 inhibition in vivo and Stb3 was recruited to both RiBi and RP-promoters upon Sch9 inhibition, which correlated with the recruitment of RPD3L [
24]. A growth defect caused by Stb3 overexpression was mitigated by the deletion of
PPH22, but more direct involvement of PP2A in the regulation of Stb3 is lacking [
98]. Stb3 is regulated via subcellular localization as glucose addition to post-log phase cells triggered its export from the nucleus within minutes, while rapamycin addition to log-phase cells had the opposite effect [
98] (
Figure 2).
Figure 2. Model of the regulation of RP and RiBi genes via the transcriptional repressors Stb3, Tod6 and Dot6 downstream of TORC1 and PKA. In the absence of phosphorylation by Sch9 and PKA, the transcription factors Stb3, Tod6 and Dot6 bind specific promoter elements upstream of a subset of RP and RiBi genes, triggering their repression via the recruitment of histone deacetylase complex RPD3L. The promoter element bound by Stb3 in RP promoters is marked with a question mark as RP promoters generally do not contain RRPE sequences and the mode of interaction with these promoters is unclear. The thickness of arrows indicates a potentially stronger relative contribution of PKA than Sch9 on Dot6 phosphorylation and vice versa for Tod6 phosphorylation. Potential dephosphorylation of Tod6 by PP2A is omitted as direct evidence is lacking.
The phosphorylation sites on Dot6, Tod6 and Stb3, which are presumably directly phosphorylated by PKA and Sch9 (and potentially dephosphorylated by PP2A), have not yet been accurately mapped. Dot6 and Tod6 are among the proteins with the highest number of PKA motifs in yeast. Mutation of six phosphorylation sites in R[R/K]x[S/T] motifs on Tod6 and four sites in R[R/K]x[S/T] and one in an Rxx[S/T] motif on Dot6 caused a decrease, but not complete abrogation of phosphorylation by Sch9 in vitro [
24]. The identity of sites phosphorylated by PKA on Dot6 and Tod6 has not been reported to date. The RRxS motif at S283 in Stb3 is conserved in yeast species from
S. cerevisiae to
C. albicans and was therefore proposed as a functionally important target in PKA signaling [
97]. Further phospho sites with PKA consensus motifs exist on Stb3 but show a lower degree of evolutionary conservation. Signal obtained from an antibody directed against the RRxS-motif on purified Stb3 was completely lost after Sch9 inhibition, indicating that Sch9 targets the same motif on this protein. Surprisingly, inhibition of PKA alone did not cause a clear change in signal [
24]. Further, the addition of cAMP to a
cyr1∆ strain was not sufficient to reverse Stb3 nuclear localization under conditions of glucose depletion or rapamycin treatment [
98]. In contrast, glucose re-addition to the same glucose-depleted strain (without cAMP) triggered Stb3 cytoplasmic localization [
98]. Therefore, cAMP neither is necessary nor sufficient for Stb3 translocation. Together, these data suggest that TOR signaling overrides PKA-dependent regulation of Stb3 under the conditions tested.
3.1.2. Ribosomal Protein Production: Ifh1, Crf1 and Spf1
The second major regulon in ribosome production are transcripts coding for ribosomal proteins (RP). As for RiBi genes, rapamycin treatment causes severe repression of this regulon [
21,
99]. The main transcription factors involved in RP gene transcription are Rap1, Hmo1 [
100], Sfp1 and Fhl1 with its co-factors Ifh1 and Crf1 [
101,
102,
103]. Of these, Rap1, Sfp1 and Fhl1-Ifh1/Crf1 have been linked to the TOR and PKA pathways [
101,
102,
104,
105,
106]. Additionally, Stb3, functions as a transcriptional repressor also of RP genes, albeit apparently not via the binding of RRPE promoter motifs [
24,
88] (
Figure 2). PKA-dependent regulation of RP genes induced by Rap1 has been shown in a number of experiments [
106,
107]: RP-transcripts were up-regulated in a Rap1-dependent manner in a
BCY1 deletion strain and the presence of the Rap1 binding site in its promoter was sufficient to confer an increase in a reporter transcript in a
bcy1∆ strain compared to wt [
108]. While phosphorylation of Rap1 via PKA cannot be completely ruled out, more recent models suggest, Rap1 serves as a recruiting factor for proteins regulated by PKA. These binding partners may include Sfp1 and Fhl1 and its co-factors [
107].
While 127 of the 138 RP genes harbor Rap1-binding sites, approximately a half are bound by Fhl1 [
101,
107]. Fhl1 is not apparently regulated by PKA or TOR, instead, regulation occurs on the level of its co-regulators Ifh1 and Crf1. The level of the Ifh1 bound to RP genes drops upon nutrient depletion or rapamycin treatment and increases when cells resume growth upon encountering improved nutrient conditions, while Fhl1 remains constitutively associated with the promoters [
101,
102,
103]. The interaction of Fhl1 and Ifh1 depends on the forkhead-associated (FHA) domain of Fhl1 [
101,
102], a domain previously reported to interact with phosphorylated sequences [
109]. It is, therefore, reasonable to posit that Fhl1-Ifh1 interaction depends on Ifh1 phosphorylation. Martin, 2004 provided a detailed description of how transcriptional activator Ifh1 and repressor Crf1 compete for binding to Fhl1 [
104]. They observed a rapamycin-triggered increase in Crf1 phosphorylation, causing its nuclear translocation and therefore RP gene repression. As this process could be prevented by mutations that render PKA signaling hyperactive, they suggested that TORC1 acts upstream of PKA in this respect [
104]. They further found that protein kinase Yak1 is required for Crf1 nuclear translocation. As Yak1 is a known direct PKA, but not TORC1/Sch9 target, this is also consistent with the idea of TORC1 regulating PKA.
Surprisingly, regulation of RP gene transcription by Crf1 appears to be yeast strain dependent, as Zhao et al. observed no effect of
CRF1 deletion on RP transcription in the W303, while they did observe the effect in the TB50 strain [
110]. The Yak1-dependent mechanism also appears to be at odds with the finding by others that RP gene transcription is still repressed by rapamycin in strains lacking Yak1 [
72]. I propose that this is explained by a secondary mechanism that acts on longer timescales. Indeed, remaining RP gene inhibition in strains lacking
CRF1 was observed also in the original study by Martin, 2004. What this second mechanism may be is not yet clear. Finally, to date, only CK2 has been shown to phosphorylate Ifh1 and Crf1 in a manner relevant for Fhl1 binding [
111,
112]. Mechanistic details if and how PKA and TORC1 regulate Fhl1-dependent RP transcription are therefore still lacking.
Sfp1 is a transcription activator of RP genes and deletion strains of SFP1 are marked by a striking small cell size phenotype [
113]. While Sfp1 binds promoters of other gene groups as well, this appears to be mechanistically different [
114].
Sfp1 was associated with TOR signaling in a microscopic screen as it localized from the nucleus to the cytoplasm upon rapamycin treatment [
115]. Importantly, PKA signaling can override TORC1 effects to some extent as hyperactivated PKA (
bcy1∆) largely counteracted the rapamycin-induced cytoplasmic relocalization. In which way do PKA and TOR signaling converge on Sfp1? Sfp1 phosphorylation was reduced upon rapamycin treatment and, in vitro phosphorylation by TORC1 was observed, while no phosphorylation was detected in an in vitro kinase assay with Sch9 [
105]. This renders Sfp1 one of only a handful of direct TORC1 substrates. Seven potential TORC1 phosphorylation sites on Sfp1 were explored and their mutation almost completely abrogated further dephosphorylation upon rapamycin treatment [
105]. Sfp1 was also phosphorylated by bovine PKA in vitro, but phosphorylation sites were not determined [
97]. An RRxS motif at a site different from the TORC1-dependent sites and conserved in multiple yeast species was proposed to constitute an important PKA site [
97]. Regulation of Sfp1 through common sites by PKA and TORC1 is therefore unlikely.